These investigations concern the role(s) of the ubiquitin system in cellular metabolism and how this system is regulated. Our focus is the ubiquitin-activating enzyme ("E1"). E1 is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. We recently found that E1 exists as two isoforms in HeLa cells (Cook and Chock, J. Biol. Chem., in press). We have designated these "E1-117 kDa" and "E1-110 kDa" to reflect their apparent molecular weights as determined by SDS-PAGE. This finding has important implications for understanding how the ubiquitin-conjugation pathway is regulated. To gain a better understanding of how these isoforms may contribute to the regulation of ubiquitin-protein conjugate formation we are studying the structural differences between the two isoforms which give rise to their different migration on SDS gels. Although several hypotheses have been tested experimentally, this structural difference remains to be identified. The most significant finding of these studies is that E1 is phosphorylated in vivo. This finding also carries implications for potential regulatory mechanisms of the pathway. In addition to these studies, we are investigating the role of ubiquitin in gene regulation. We have purified the ubiquitin-conjugating enzymes, E1, E2, and E3, and have tested the specificity of the ubiquitin- conjugating enzymes using two different recombinant gene-regulatory proteins (c-fos and c-jun). We have also measured the effect of ubiquitination on the functional properties of these gene regulatory proteins.